Mycobacterium tuberculosis possesses a cell envelope that contributes to this bacterium's innate resistance to many antibiotics, and its ability to cause disease (tuberculosis) and survive in the presence of the host's immune response. This envelope is comprised of the mycolic acid-arabinogalactan-peptidoglycan (mAGP) cell wall core where the mycolic acids are oriented toward the surface of the cell. Further, an outer layer of lipids and lipoglycans form a bilayer with the covalently linked mycolic acids of mAGP. Our recent studies of the lipids and mycolic acids produced by M. tuberculosis during infection of the guinea pig lung revealed a dramatic shift in the lipid profile as compared to that of the bacterium grown in vitro. Two cell envelope products, phthiocerol dimycocerosic acids (PDIM) and mycolic acids were substantially altered when produced during in vivo growth and both are known to contribute to pathogenesis. Specifically, the mycocerosic acids of the PDIM significantly increased in length; and the ratio of 1-, keto- and methoxymycolic acids shifted such that a previously unrecognized form of the M. tuberculosis 1-mycolate became the dominant mycolic acid species of in vivo grown bacilli. Stationary phase in vitro cultures of M. tuberculosis produced in vivo like PDIMs but not in vivo like mycolic acid profiles. These data demonstrated differential regulation in the biosynthesis of these two cell envelope products. Understanding the basic regulatory events that alter the in vivo profile of PDIM and mycolic acids will allow for future studies that target the role of in vivo lipid structures in host-pathogen interactions. Manipulation of these regulatory mechanisms also will allow for development of methods to screen potential anti-tuberculosis drugs against cells with a phenotype that more accurately represents the bacterium in vivo. This R21 application we will provide information on regulatory mechanisms that control mycocerosic acids chain length (Specific Aim 1); and that are responsible for the shift between in vitro and in vivo mycolic acid profiles (Specific Aim 2).